Formulations containing soluble gp130 dimers and methods of use - Patent Application 20070122997

Abstract

The present invention provides an aqueous formulation comprising a dimer of two single-chain gp130-Fc fusion proteins, a histidine salt, trehalose, and polysorbate 80. The aqueous formulation can be used to treat an inflammatory disease or an IL-6 mediated disease.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine research, and specifically relates to a formulation containing gp130 dimer and its use for treating various IL-6-mediated diseases, including inflammatory diseases and cancer. [Background technology]

[0002] Glycoprotein 130 (also known as gp130, IL6ST, IL6-beta, or CD130) is a transmembrane protein. It forms one subunit of the type I cytokine receptor in the IL-6 receptor family. It is crucial for signal transduction after cytokine engagement. Structurally, the extracellular portion of gp130 consists of five fibronectin type III domains and one immunoglobulin-like C2 domain.

[0003] All members of the IL-6 receptor family transmit signals by forming complexes with gp130. For example, IL-6 binds to the IL-6 receptor. The complex of these two proteins then associates with gp130. The three-protein complex then dimerizes to form a hexameric complex that can generate downstream signals.

[0004] IL-6 is a multifunctional cytokine produced by hematopoietic and non-hematopoietic cells, e.g., in response to infection and tissue injury. IL-6 exerts its multiple biological activities via two major signaling pathways: the so-called classical ligand-receptor pathway, which binds to the IL-6R via the membrane, primarily present in hepatocytes and some leukocytes; and the cross-signaling pathway (trans-signaling pathway) via the circulating sIL-6R (soluble IL-6R), which is derived from the membrane-bound IL-6R by proteolytic cleavage or alternative splicing.

[0005] In the classical pathway, IL-6 binds directly to the membrane-bound IL-6R on the surface of a limited range of cell types. The IL-6 / IL-6R complex associates with a preformed dimer of the signal-transducing receptor protein gp130, triggering spatial changes in the gp130 dimer, thereby initiating an intracellular signaling cascade. Classical signaling is responsible for acute inflammatory defense mechanisms and important physiological IL-6 functions, such as signaling for growth and regeneration in intestinal epithelial cells. The extracellular domains of IL-6R and gp130 can be generated by translating alternatively spliced mRNAs without the membrane-anchoring domain, generating sIL-6R and gp130 variants.

[0006] The activity of the IL-6 / sIL-6R complex is generally controlled by high levels of soluble sgp130 present in the circulation, which effectively competes with membrane-bound gp130. The gp130 dimer of the present invention has a higher binding affinity than native sgp130 and is therefore more potent at inhibiting IL-6 signaling. The formulation of the present invention allows for more stable production, transport, and operation of the gp130 dimer. Summary of the Invention

[0007] To overcome the problems in the prior art, the present invention provides a formulation containing a gp130 dimer (or a "gp130-containing fusion protein," or simply "fusion protein") and its use for treating various IL-6-mediated diseases, including inflammatory diseases and cancer. The formulation contains a histidine salt buffer system, has a pH of about 7.6, is highly stable, and can be safely administered to humans at various doses.

[0008] In one embodiment, the present specification describes an aqueous formulation (which may also be referred to as a liquid formulation) and a lyophilized formulation, which comprises two monomers having an amino acid sequence of SEQ ID NO: 1, and a fusion protein linked by a plurality of disulfide bonds, 20 to 30 mM histidine salt, 220 to 280 mM trehalose, 0.01 (w / v)% to 0.03 (w / v)% polysorbate 80, and its pH is 7.0 to 8.2. In some embodiments, it comprises at least 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or at least 30 mg / mL of the fusion protein.

[0009] In some embodiments, the pH of the aqueous formulation is 7.4 to 7.8. In some embodiments, the pH of the aqueous formulation is 7.6.

[0010] In some embodiments, the aqueous formulation comprises 24 to 26 mM histidine salt. In some embodiments, the aqueous formulation comprises 25 mM histidine salt.

[0011] In some embodiments, the aqueous formulation comprises 240 to 260 mM trehalose. In some embodiments, the aqueous formulation comprises 250 mM trehalose.

[0012] In some embodiments, the aqueous formulation comprises 0.015 (w / v)% to 0.025 (w / v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02 (w / v)% polysorbate 80.

[0013] In some embodiments, the aqueous formulation further comprises an isotonic agent (or osmotic pressure regulator or stabilizer), a surfactant, an antioxidant, a preservative, or a mixture thereof.

[0014] In some embodiments, each fusion protein molecule has 6 or fewer galactose-α-1,3-galactose PartIn some embodiments, each fusion protein molecule comprises no more than three, two, or one galactose-α-1,3-galactose. Part Includes:

[0015] In some embodiments, the fusion protein comprises glycans, wherein an average of at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein an average of at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein an average of 52-65% of the glycans comprise one or more sialic acid residues.

[0016] In one embodiment, the aqueous formulation comprises at least 25 mg / mL of the fusion protein, 24 to 26 mM histidine salt, 240 to 260 mM trehalose, and 0.015 (w / v)% to 0.025 (w / v)% polysorbate 80, and has a pH of 7.6 to 7.8.

[0017] In one preferred embodiment, the aqueous formulation contains 30 mg / mL of the fusion protein, 25 mM histidine salt, 250 mM trehalose, and 0.02 (w / v)% polysorbate 80, and has a pH of 7.6.

[0018] In some embodiments, the aqueous formulation contains no other amino acid salts other than histidine salts at greater than 10 mM (or no greater than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM), or preferably no other amino acid salts at all.

[0019] In some embodiments, the aqueous formulation contains no other sugars other than trehalose at greater than 10 mM (or greater than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM), or preferably no other sugars at all.

[0020] In some embodiments, the aqueous formulation is for treating an inflammatory disease or IL-6-mediated disease in a human. In some embodiments, the inflammatory disease or IL-6-mediated disease is inflammatory bowel disease, preferably wherein the treatment induces remission of the inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis, preferably wherein the treatment maintains remission of the inflammatory bowel disease. In some embodiments, the inflammatory disease or IL-6-mediated disease is rheumatoid arthritis, psoriasis, uveitis, or atherosclerosis. In some embodiments, the inflammatory disease or IL-6-mediated disease is colitis unrelated to inflammatory bowel disease, preferably wherein the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis, or colitis resulting from an allergy affecting the large intestine.

[0021] This specification describes dry formulations, which can be obtained by lyophilizing any one of the aqueous formulations described herein, or which can be produced upon addition of water to any one of the aqueous formulations described herein. [Brief explanation of the drawings]

[0022] [Figure 1] This shows the results of pH / buffer system screening DSC. [Figure 2] This shows the results of pH / buffer system screening HT-DLS. [Figure 3] 1 shows the results of DSC screening of auxiliary materials and surfactants. [Figure 4] 1 shows the appearance of freeze-dried products with different formulations. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition All numerical symbols (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximate values that change in increments of 0.1 or 10% (1) or (-). It should be understood that, although not necessarily explicitly stated, the term "about" precedes all numerical symbols. Further, it should be understood that, although not necessarily explicitly stated, the reagents described herein are exemplary and their equivalents are known to those skilled in the art.

[0024] The terms "protein" and "polypeptide" can be used interchangeably and, in their broadest sense, refer to compounds of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be joined by peptide bonds. In another embodiment, the subunits may be joined by other bonds such as, for example, esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids with respect to the sequences that can constitute a protein or peptide. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine, D and L optical isomers, amino acid analogs, and peptidomimetics. The one-letter and three-letter abbreviations for the naturally occurring amino acids are shown below.

[0025] "Composition" is intended to mean a combination of an active agent with other inert agents (e.g., detectable reagents or labels) or active compounds or compositions (e.g., adjuvants). "Pharmaceutical composition" is intended to include a combination of an active agent with an inert or active carrier to produce a composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0026] "Aqueous formulation" is a liquid formulation that uses water as a solvent. In one embodiment, the aqueous formulation is a formulation that does not require lyophilization, spray drying, and / or freezing to maintain stability (e.g., chemical and / or physical stability and / or biological activity).

[0027] As used herein, the term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Pharmaceutically acceptable buffers include, but are not limited to, tris buffer, arginine buffer, histidine buffer, citrate buffer, succinate buffer, and phosphate buffer. Regardless of the buffer used, the pH may be adjusted with acids or alkalis known in the art, such as succinic acid, hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, succinate, citrate, tris base, histidine, histidine HCl, sodium hydroxide, and potassium hydroxide. Suitable buffers include, but are not limited to, histidine buffer, 2-morpholinoethanesulfonic acid (MES), dimethylarsinate, phosphate, acetate, succinate, and citrate. The concentration of the buffer may be between 4 mM and about 60 mM, or alternatively between about 4 mM and about 40 mM, or alternatively between about 5 mM and about 25 mM.

[0028] As used herein, the terms "treatment", "therapy", and "treatment" refer to reversing a disease or disorder described herein or one or more symptoms thereof, reducing a disease or disorder or one or more symptoms thereof, delaying the onset of a disease or disorder or one or more symptoms thereof, or suppressing the progression of a disease or disorder or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have progressed. In other embodiments, treatment may be administered asymptomatically. For example, treatment can be administered to a susceptible individual before symptoms develop (e.g., taking into account a symptom history and / or genetic or other susceptibility factors). Treatment, such as prevention or delay of recurrence, can also continue after symptoms have resolved.

[0029] The interpretation and examples of proper nouns or terms in each theme of the present invention are all common and will not be described in detail. Formulation

[0030] IM001 is a dimer containing two single-chain gp130-Fc fusion proteins. It may be used to treat various IL-6-mediated diseases, including inflammatory diseases and cancer. Like other protein drugs, the solubility, stability, and activity of IM001 are affected by its environment. Therefore, developing an appropriate formulation with an appropriate buffer system is challenging.

[0031] The inventors prepared 12 pH / buffer system formulations (Table 2) and nine supplementary and surfactant aqueous formulation screening formulations (Table 7). Stability was examined at 30°C for two weeks and compared using DSC, DLS, appearance, protein concentration, pH, SEC-HPLC, and SDS (reduced / non-reduced). At pH ≤ 6.5, visible foreign matter was clearly observed and the protein was unstable. However, a buffer system at pH 8.0 (slightly alkaline) was found to be more stable. Furthermore, for the same pH 8.0 buffer system, the histidine buffer system exhibited greater stability than the glycine and Tris buffer systems. Interestingly, the addition of IM001 protein to the buffer system caused a pH drift. In the pH 8.0 histidine buffer system, the pH drifted to 7.6 upon protein addition.

[0032] After considering various aqueous formulations, the inventors of the present application found that the optimum stability was achieved at a protein concentration of 15 mg / mL under the protective effects of sucrose and polysorbate 80. Unfortunately, however, the stability was not sufficient when the protein concentration reached 30 mg / mL.

[0033] This result is surprising because many proteins containing Fc fragments, such as antibodies, can be easily formulated into stable aqueous formulations at higher concentrations. Without being bound by any particular theory, the inventors of the present application believe that this is because the molecular properties of the fusion protein make it prone to instability at high temperatures or high concentrations, and the isoelectric point limits the pH buffer systems that can be selected, making it more challenging to develop formulations for general biological drug molecules such as mabs.

[0034] To improve the stability of high-concentration formulations, the inventors of this application prepared five lyophilized formulation screening formulas (Table 14) and examined their stability at 25°C and 40°C. Experimental results showed that at the same high concentration of 30 mg / mL, the stability of the lyophilized product was significantly improved compared to the solution formulation. Interestingly, the lyophilized formulation (30 mg / mL, 25 mM His, 250 mM Trehalose, 0.02% PS80, pH 7.6) performed well at various temperatures and in various tests, with little change even after two months at a high temperature of 40°C. This result was quite surprising, as the same formulation generally does not necessarily have advantages in various tests. Therefore, all of these formulations were used as lyophilized formulations of IM001 protein. Furthermore, the sugar (trehalose) used in this lyophilized formulation differs from the sugar (sucrose) preferred in aqueous formulations.

[0035] Based on these test results, the present application provides aqueous and lyophilized formulations applicable to IM001, which include a fusion protein, a histidine salt, trehalose, and a polysorbate. In some embodiments, the aqueous formulation can be lyophilized to form a lyophilized formulation. In some embodiments, the lyophilized formulation can be added with appropriate water to produce the aqueous formulation. Such aqueous formulations can also be injected into patients to treat the indicated diseases.

[0036] As previously mentioned, the fusion protein (IM001) herein comprises two monomers having the amino acid sequence of SEQ ID NO: 1, linked by multiple disulfide bonds. In some embodiments, each fusion protein molecule contains six or fewer galactose-α-1,3-galactose residues. Part In some embodiments, each fusion protein molecule comprises no more than three, two, or one galactose-α-1,3-galactose. PartIn some embodiments, the fusion protein comprises glycans, wherein an average of at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein an average of at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein an average of 52-65% of the glycans comprise one or more sialic acid residues.

[0037] In some embodiments, the aqueous formulation contains at least 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or at least 30 mg / mL of the fusion protein, hi some embodiments, the aqueous formulation contains 10-60 mg / mL, 15-45 mg / mL, 20-40 mg / mL, 25-35 mg / mL, or 30 mg / mL of the fusion protein.

[0038] In some embodiments, the aqueous formulation comprises at least 10 mM histidine salt. In some embodiments, the aqueous formulation comprises at least 15 mM, 20 mM, 25 mM, 30 mM, or 35 mM histidine salt. In some embodiments, the aqueous formulation comprises 10-50 mM histidine salt, or 10-40 mM, 15-35 mM, 20-30 mM, 22-28 mM, or 24-26 mM histidine salt. In some embodiments, the aqueous formulation comprises 25 mM histidine salt.

[0039] In some embodiments, the aqueous formulation contains at least 100 mM trehalose. In some embodiments, the aqueous formulation contains at least 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM trehalose. In some embodiments, the aqueous formulation contains 100-400 mM trehalose, or 150-350 mM, 200-300 mM, 220-280 mM, 240-260 mM, or 245-255 mM trehalose. In some embodiments, the aqueous formulation contains 250 mM trehalose.

[0040] In some embodiments, the aqueous formulation comprises a polysorbate, such as polysorbate 20 or polysorbate 80. In some embodiments, the aqueous formulation comprises at least 0.005% (w / v) polysorbate. In some embodiments, the aqueous formulation comprises at least 0.01% (w / v) polysorbate, or at least 0.015% (w / v) polysorbate, or at least 0.02% (w / v) polysorbate, or at least 0.025% (w / v) polysorbate. In some embodiments, the aqueous formulation comprises 0.01% (w / v) to 0.03% (w / v) polysorbate 80. In some embodiments, the aqueous formulation comprises 0.015% (w / v) to 0.025% (w / v) polysorbate 80. In some embodiments, the aqueous formulation comprises 0.018% (w / v) to 0.022% (w / v) polysorbate 80. In some embodiments, the aqueous formulation comprises 0.019% (w / v) to 0.021% (w / v) polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02% (w / v) polysorbate 80.

[0041] In some embodiments, the pH of the aqueous formulation is 7.0 or higher. In some embodiments, the pH of the aqueous formulation is 7.1 or higher, 7.2 or higher, 7.3 or higher, 7.4 or higher, 7.5 or higher, or 7.6 or higher. In some embodiments, the pH of the aqueous formulation is 7.0 to 8.2 or 7.1 to 8.1, 7.2 to 8.0, 7.3 to 7.9, 7.4 to 7.8, 7.5 to 7.7, or 7.55 to 7.65. In some embodiments, the pH of the aqueous formulation is 7.6.

[0042] In one exemplary embodiment, the aqueous formulation comprises at least 25 mg / mL of the fusion protein, 24 to 26 mM of a histidine salt, 240 to 260 mM of trehalose, and 0.015 (w / v)% to 0.025 (w / v)% of polysorbate 80, and its pH is 7.4 to 7.8. In one exemplary embodiment, the aqueous formulation consists of at least 25 mg / mL of the fusion protein, 24 to 26 mM of a histidine salt, 240 to 260 mM of trehalose, and 0.015 (w / v)% to 0.025 (w / v)% of polysorbate 80, and its pH is 7.4 to 7.8.

[0043] In one exemplary embodiment, the aqueous formulation comprises at least 25 mg / mL of the fusion protein, 25 mM of a histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and its pH is 7.6. In one exemplary embodiment, the aqueous formulation consists of 30 mg / mL of the fusion protein, 25 mM of a histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and its pH is 7.6.

[0044] In some embodiments, the aqueous formulation further comprises an isotonic agent (or osmotic pressure regulator, also called a stabilizer), a surfactant, an antioxidant, a preservative, or a mixture thereof.

[0045] In some embodiments, the aqueous formulation further comprises a tonicity agent (also called an osmotic pressure adjuster or stabilizer). As used herein, the term "tonicity agent" refers to a pharmaceutically acceptable agent for adjusting the tonicity of a formulation. Isotonicity generally relates to the osmotic pressure of a solution, generally relative to human serum. A formulation may be reduced in tonicity, isotonic, or increased in tonicity. Alternatively, the formulation may be isotonic. An isotonic formulation refers to a solution that is reconstituted from a liquid or solid form (e.g., a lyophilized form) and has the same tonicity as some other solution (e.g., saline solution and serum) compared to it. Suitable isotonicity agents include, but are not limited to, sodium chloride, potassium chloride, glycerin, mannitol, amino acids as defined herein, any sugar-derived component, and combinations thereof.

[0046] In some embodiments, the aqueous formulation further comprises a surfactant. As used herein, the term "surfactant" refers to a pharmaceutically acceptable organic substance with an amphiphilic structure, i.e., it is composed of a group with reverse solubility, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. The charge of the visible surface-active moiety of surfactants can be classified as anionic, cationic, or nonionic. Surfactants are commonly used as wetting agents, emulsifiers, solubilizers, and dispersants in many pharmaceutical compositions and biomaterial formulations. In some embodiments of the pharmaceutical formulations described herein, the amount of surfactant is expressed as a weight / volume percentage (w / v%). Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene diluted ethers, diluted phenyl polyoxyethylene ethers (Triton-X), polyoxyethylene diluted-polyoxypropylene copolymers (poloxamers, Pluronic), or sodium dodecyl sulfate (SDS). Polyoxyethylene sorbitan fatty acid esters include polysorbitan ester 20 (trademark Tween 20) TM (sold under the trademark Tween 80) and Polysorbitan Ester 80 TMincluding those sold under). The polyethylene - polypropylene copolymer includes Pliironic(R) F68 or poloxamer 188 TM including those sold under that name. The polyoxyethylene alkyl ether includes those sold under the trademark Bri j TM including those sold under. The alkylphenol polyoxyethylene ether includes those sold under the trade name Triton - X.

[0047] In some embodiments, the aqueous formulation further includes an antioxidant. An "antioxidant" refers to a molecule that can retard or prevent the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. The oxidation reaction can generate radicals that initiate a chain reaction that destabilizes protein therapeutics and ultimately affects the activity of the product. Antioxidants stop these chain reactions by removing radical intermediates and suppress other oxidation reactions by oxidizing themselves. Therefore, antioxidants are generally reducing agents, chelating agents, and deoxidizers such as citrate, EDTA, DPTA, thiols, ascorbic acid, or polyphenols. Non - limiting examples of antioxidants include ascorbic acid (AA, E300), thiosulfate, methionine, tocopherol (E306), propyl gallate (PG, E310), tert - butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA, E320), butylated hydroxytoluene (BHT, E321).

[0048] In some embodiments, the aqueous formulation further comprises a preservative. A "preservative" is a natural or synthetic chemical that is added to products such as food, pharmaceuticals, paints, biological samples, wood, etc. to prevent the growth of microorganisms or decomposition due to unwanted chemical changes. Preservative additives can be used alone or in combination with other preservation methods. The preservative may be an antibacterial preservative that inhibits the growth of bacteria and fungi, an antioxidant such as an oxygen scavenger that inhibits the oxidation of components. Common antibacterial preservatives include benzalkonium chloride, benzoic acid, chlorhexidine, glycerin, benzoic acid, potassium sorbate, chili oil, sulfites (such as sulfur dioxide, sodium bisulfite, potassium bisulfite), and disodium EDTA. Other preservatives include those commonly used for parenteral proteins, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, methyl p-hydroxybenzoate.

[0049] As can be seen from the examples, some commonly used biological pharmaceutical formulation aids, such as glycine, Tris, mannitol, etc., do not contribute to the formation of excellent aqueous or lyophilized formulations. Therefore, in some embodiments, the aqueous formulation does not contain these auxiliary materials.

[0050] In some embodiments, the aqueous formulation does not contain amino acid salts other than histidine salts that are higher than 10 mM (or do not include those higher than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM), such as arginine, lysine, asparagine, glutamine, glycine, or their salts. In some embodiments, the aqueous formulation does not contain amino acid salts other than histidine salts, such as arginine, lysine, asparagine, glutamine, glycine, or their salts.

[0051] In some embodiments, the aqueous formulation does not contain sugars other than trehalose that are higher than 10 mM (or not higher than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM), such as sucrose. In some embodiments, the aqueous formulation does not contain sugars other than trehalose, such as sucrose.

[0052] In some embodiments, the present specification describes corresponding dry formulations, such as lyophilized formulations. In some embodiments, the dry formulation is obtained by lyophilizing the aqueous formulation described herein. In some embodiments, the dry formulation can produce the aqueous formulation described herein when an appropriate amount of water is added. Use

[0053] The formulations described in the present application can be used for the treatment of various corresponding diseases. The gp130 dimer in the present invention has a higher binding affinity than native soluble gpl30, and thus has a stronger ability to suppress IL-6 signaling. IL-6 signaling is associated with many diseases, including the diseases briefly described below and other diseases generally known to those skilled in the art.

[0054] Chronic inflammation, such as Crohn's disease (CD), ulcerative colitis (UC), rheumatoid arthritis (RA) or psoriasis, is histologically related to the presence of monocytes such as macrophages and lymphocytes, and persists in tissues even after being obtained for the resolution of the acute inflammatory phase. In chronic inflammatory disease models, IL-6 appears to have a harmful effect of promoting monocyte accumulation at the site of injury by inducing continuous MCP-I secretion, angiogenesis, and anti-apoptotic functions in T cells.

[0055] Inflammatory bowel disease (IBD), namely Crohn's disease (CD) or ulcerative colitis (UC), is a chronic inflammation that occurs in the intestinal tract of susceptible individuals and is thought to be independent of specific pathogens. Changes in the epithelial mucosal barrier lead to enhanced mucosal immune system associated with increased intestinal permeability, and exposure to intestinal antigens results in inappropriate activation of the patient's intestinal immune system. Uncontrolled activation of mucosal CD4+ T-lymphocytes is accompanied by continuous over-release of pro-inflammatory cytokines, inducing pathogenic gastrointestinal inflammation and tissue damage. There is a common understanding that the main activated immune cells involved in the pathogenesis of IBD are intestinal T cells and macrophages.

[0056] IL-6 has been shown as a central cytokine in IBD in humans. It was found that CD and UC patients produced elevated levels of IL-6 compared to the control group, and that IL-6 levels were associated with clinical activity. It was also found that the level of the IL-6 / sIL-6R complex in serum increased due to the increase in the level of sIL-6R in (3) patients. Lamina propria mononuclear cells obtained from surgical colon specimens of CD and UC patients showed increased production of IL-6 by both CD4+ T cells and macrophages compared to controls. It was found that sIL-6R was shed and released from the surface of macrophages and monocytes with the increased production associated with the increase in IL-6 levels. In CD patients, mucosal T cells showed strong evidence of IL-6 cross-signaling, accompanied by activation of STAT3, bcl-2, and bcl-xl. Blockade of IL-6 cross-signaling induced apoptosis of T cells, indicating that the IL-6 / sIL-6R system mediates resistance to apoptosis of T cells in CD.

[0057] Therefore, in IBD patients, the acquired accumulation of pro-inflammatory CD4+ T cells in the lamina propria, which leads to the persistent presence of inflammation, is highly dependent on anti-apoptotic IL-6 / sIL-6R cross-signaling. The polypeptides disclosed herein are thought to be able to be used for the treatment of CD and other inflammatory diseases by acting on the IL-6 / sIL-6R complex.

[0058] Therefore, the formulations of the present invention are capable of treating IL-6 mediated disorders. IL-6 mediated disorders include inflammatory diseases or cancer. In this aspect, the polypeptides and compositions described herein can be administered to subjects suffering from an inflammatory disease, such as, for example, juvenile idiopathic arthritis, Crohn's disease, colitis (e.g., radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis not related to inflammatory bowel disease (IBD) caused by allergies affecting the colon), dermatitis, psoriasis, uveitis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In some embodiments, the inflammatory disease is selected from the group consisting of diabetes, gout, cryoglobulin-related periodic syndromes and chronic obstructive pulmonary disease.

[0059] Preferably, the inflammatory disease or IL-6 mediated disorder is an inflammatory bowel disease, and preferably here, the treatment induces remission of the inflammatory bowel disease. Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis, and preferably here, the treatment maintains remission of the inflammatory bowel disease. Preferably, the inflammatory disease or IL-6 mediated disorder is rheumatoid arthritis, psoriasis, uveitis or atherosclerosis. Preferably, the inflammatory disease or IL-6 mediated disorder is colitis not related to inflammatory bowel disease, and preferably here, the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis caused by allergies affecting the colon. Preferably, the inflammatory disease or IL-6 mediated disorder is selected from Crohn's disease, ulcerative colitis, rheumatoid arthritis and psoriasis, and more preferably, selected from Crohn's disease and ulcerative colitis.

[0060] In the case of an inflammatory disease such as an inflammatory bowel disease, the treatment can include alleviation of symptoms, maintenance of symptom alleviation, or both.

[0061] In other embodiments, provided is a method for treating cancer, reducing the severity of cancer, or preventing cancer, where the cancer includes, but is not limited to, multiple myeloma, plasma cell leukemia, renal cell carcinoma, Kaposi's sarcoma, colorectal cancer, gastric cancer, melanoma, leukemia, lymphoma, glioma, glioblastoma multiforme, lung cancer (including, but not limited to, non-small cell lung cancer (NSCLC, adenocarcinoma and squamous cell carcinoma)), non-Hodgkin lymphoma, Hodgkin lymphoma, plasmacytoma, sarcoma, thymoma, breast cancer, prostate cancer, hepatocellular carcinoma, bladder cancer, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, esophageal cancer, liver cancer, ovarian cancer, acute lymphoblastic leukemia (ALL), T-ALL, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), salivary gland cancer or other cancers.

[0062] In another embodiment of the present disclosure, provided is a method for treating a disease, reducing the severity of the disease, or preventing the disease, where the disease is selected from the group consisting of sepsis, bone resorption disease (osteoporosis), cachexia, cancer-related fatigue, psoriasis, systemic juvenile idiopathic arthritis, systemic lupus erythematosus (SLE), mesangial proliferative glomerulonephritis, hypergammaglobulinemia, Castleman disease, IgM gammaglobulinemia, cardiac myxoma and autoimmune insulin-dependent diabetes. Production method

[0063] In a further aspect of the present disclosure, a production method of the formulation is provided. The cDNA encoding SEQ ID NO:1 may be cloned into a vector such that the signal peptide binds within the amino-terminal frame of the amino acid sequence of the antibody chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from non-immunoglobulin).

[0064] The design of an expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed and the expression level of the desired protein. Regulatory sequences for expression in mammalian host cells include viral elements that induce high-level protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), promoters and / or enhancers derived from polyomavirus and strong mammalian promoters (e.g., native immunoglobulin and actin promoters). The host cell may be a mammalian, insect, plant, bacterial, or yeast cell, and preferably, the cell is a mammalian cell, such as Chinese hamster ovary (CHO) cells. Exemplary CHO cells are those obtained from the European Collection of Cell Cultures (ECACC, accession number 9406067) ((CHO) / dhfr - cells. Preferably, the host cell is a CHO cell, and the nucleic acid encoding the polypeptide is codon-optimized for use in CHO cells.

[0065] Another aspect of the disclosure includes a fusion protein produced by the methods disclosed herein. Preferably, the dimer has the characteristics described herein (e.g., % galactose-α-1,3-galactose per mole of polypeptide Part , sialylation). The dimer produced by the method may be used to produce an appropriate composition. The fusion protein molecule produced in this way contains 6 or fewer galactose-α-1,3-galactose Part , or 3, 2, or 1 or fewer galactose-α-1,3-galactose Part .

[0066] The fusion protein molecules produced in this way contain glycans, and on average at least 52% of said glycans contain one or more sialic acid residues. In some embodiments, said fusion protein contains glycans, and on average at least 54% of said glycans contain one or more sialic acid residues. In some embodiments, said fusion protein contains glycans, where on average 52 - 65% of said glycans contain one or more sialic acid residues.

Example

[0067] Example 1 The IM001 protein is obtained from CHO-K1 modified cells expressing the gp130-Fc fusion protein gene through cell culture, isolation, and purification.

Table 1

[0068] The cDNA sequence of IM001 is expressed in the CHO cell expression system. Due to the presence of the IgGl Cys-Pro-Pro-Cys sequence in the Fc region, two identical gp130-Fc subunits are dimerized by the mercapto residues on the Fc region, and together they form IM001.

[0069] The test production and purification process of the IM001 drug substance is as follows. Before inoculating the production bioreactor, the cells were revived from the WCB vial using protein-free medium and gradually expanded. After the cell culture was completed, cells and cell debris were removed by culture filtration. The purification consists of three column chromatography steps, one concentration and elution step, and two specific virus removal steps (virus inactivation treatment and nanofiltration, removal of enveloped and non-enveloped viruses). After concentration and elution, excipients were added to prepare the drug substance. The prepared IM001 was filtered into a container through a 0.22 μm filtration membrane.

[0070] This example also attempted to screen the most stable pH / buffer system of the IM001 protein.

[0071] The IM001 stock solution was exchanged with 20 mM acetate (pH 4.5, pH 5.0, pH 5.5), citrate (pH 5.0), histidine-aspartate (pH 5.0, pH 5.5), histidine (pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 8.0), and phosphate (pH 7.0) by dialysis method. The protein concentration was adjusted to about 30 mg / mL and filtered through a 0.22 μm PVDF membrane filter. After filtration, the samples were bottled and sealed. All operations were performed inside a biosafety cabinet. One of them was designated as T0, appropriate stability consideration conditions were selected based on the results of T0 DSC and HT-DLS, the remaining samples were stored under these conditions, and sampling analysis was performed at the time points specified in Table 2 for each one. [Table 2] 1.1. DSC and HT-DLS Results

[0072] The details of the results are shown in Table 3 and Figures 1 - 2. The Tm-onset of all formulations was 40.9 - 43.0 °C, and the Tagg Onset, except for Formulation 1 at 60.6 °C and Formulation 11 at 65.7 °C, was about 45 °C for the other formulations. [Table 3] 1.2. Results of Appearance, pH, and Protein Concentration

[0073] The details of the results are shown in Table 4. According to the appearance results, visible foreign matters appeared in all formulations except Formulation 1 and Formulation 11 which had no visible foreign matters at T0. After storage at 30 °C for 1 week and 2 weeks, visible foreign matters appeared in all formulations. The results of pH and protein concentration showed that there were no significant changes in all formulations even after storage at 30 °C for 2 weeks. [Table 4] 1.3. SEC Purity Results

[0074] The detailed results are shown in Table 5. The results show that after storage at 30°C for 1 to 2 weeks, the SEC purity of all formulations decreased to different degrees, with formulation 11 showing the lowest decrease (approximately 13%), and the decrease ranges for the other buffer systems ranging from 19.3% to 72%. [Table 5] 1.4. SDS-PAGE (reducing / non-reducing) results

[0075] The detailed results are shown in Table 6. According to the results of reducing SDS-PAGE, after 2 weeks of storage at 30°C, the purity of p10 (His at pH 7.0), p11 (His at pH 8.0), and p12 (Phosphate at pH 7.0) did not decrease, while the purity of the remaining samples all decreased to different degrees, with the decrease ranging from 10.8% to 50.0%. The purity of non-reducing SDS-PAGE also decreased to different degrees, with the decrease of p10 (His at pH 7.0), p11 (His at pH 8.0), and p12 (Phosphate at pH 7.0) being smaller than that of the remaining samples, with the decrease ranging from 14.6% to 65.7%. [Table 6]

[0076] In this study, the most stable pH / buffer system of IM001 protein was screened by DSC, DLS, appearance, protein concentration, pH, SEC-HPLC, and SDS-PAGE (reducing / non-reducing) methods. According to the results of DSC and DLS, the Tm Onset and Tagg Onset of IM001 molecules showed an upward trend with the increase of pH value. Among them, the Tm Onset and Tagg Onset of p11 (His at pH 8.0) were higher than those of the remaining samples. After observing for 2 weeks at 30 °C, according to the results of appearance, SEC-HPLC, and SDS-PAGE (reducing / non-reducing), IM001 was unstable in a low pH environment, and formulation 11 (histidine salt buffer system, pH 8.0, protein-containing pH 7.6) was superior to other formulations in each test. Example 2

[0077] The research objective of this example is to screen appropriate auxiliary material stabilizers based on pH / buffer system screening.

[0078] Based on the results of the pH / buffer system screening experiment, a buffer system mainly evaluated with 25 mM histidine buffer system at pH 8.0 as the auxiliary material was screened. In addition, glycine buffer system at pH 7.5 and Tris buffer system at pH 7.5 were added for consideration. The IM001 stock solution was exchanged into 25 mM histidine buffer system (pH 8.0), 25 mM glycine buffer system (pH 7.5), and 25 mM Tris buffer system (pH 7.5) by ultrafiltration centrifugation method. Different auxiliary material mother liquors and surfactant mother liquors were added based on the experimental protocol, and the protein content was adjusted to about 30 mg / mL and 15 mg / mL respectively. Finally, nine formulations containing different auxiliary materials and surfactants were prepared. After filtering with a 0.22 μm PVDF membrane in a biosafety cabinet respectively, they were dispensed into vials, stoppered, and sealed. The experimental investigations under different conditions were started, and the specific protocol is shown in Table 7. The formulation pH in the table is the buffer pH before adding the protein. Since there is a pH drift phenomenon when IM001 protein is added to the buffer system, the actual formulation pH is shown in the measurement results.

Table 7

[0079] After preparing all the formulation samples, DSC was performed on the samples. The results are shown in Table 8, and the DSC curves are shown in Figure 3. The data show that the Tm Onset of the nine samples was around 45°C, and there was no significant difference between the Tm Onsets of the different formulation samples. [Table 8] 2.2 Visual detection results of auxiliary materials and surfactant screening experiments

[0080] The appearance results of the auxiliary material and surfactant screening experiments are summarized in Table 9. According to the data, after five cycles of freeze-thawing, no visible particles were observed in any of the nine formulation samples. However, after shaking at 25°C and 300 rpm for seven days, many visible particles appeared in all nine formulation samples. After two weeks of observation under high temperature (30±2°C) conditions, visible particles appeared in formulations F1 and F4. After one month of observation under accelerated conditions (25±2°C), visible particles appeared in formulations F1, F4, and F5. After one month of observation under long-term conditions (2-8°C), visible particles appeared in all formulations except for formulation F5. [Table 9] 2.3 Osmolarity, pH, and protein concentration results of auxiliary materials and surfactant screening experiments

[0081] The results of the osmotic pressure, pH and protein concentration of the auxiliary material and surfactant screening experiments are summarized in Table 10. The data show that the pH and protein concentration of the nine formulation samples did not change significantly after repeated freezing and thawing, shaking, high temperature (30±2°C) examination, accelerated temperature (25±2°C) and 2-8°C examination. [Table 10] 2.4 Results of Insoluble Particle Detection (MFI) in the Screening Experiment of Auxiliary Materials and Surfactants

[0082] The results of insoluble particle detection (MFI) in the screening experiment of auxiliary materials and surfactants are summarized in Table 11. According to the data of freeze-thaw samples, the number of insoluble particles in the F1 formulation sample was more than that of the remaining samples after freeze-thaw. According to the sample data investigated at 30 °C for 2 weeks, the number of insoluble particles in the F1 formulation sample was more than that of the remaining samples. Since there were many visible particles in the F4 formulation sample, MFI detection was not carried out. According to the data of samples investigated at 25 °C for 1 month, the number of insoluble particles in the F1 and F4 formulation samples was higher than that of the remaining samples. [Table 11] 2.5 Results of Purity (SEC-HPLC) in the Screening Experiment of Auxiliary Materials and Surfactants

[0083] The SEC-HPLC detection results are summarized in Table 12. According to the data of samples investigated at 25 °C for 1 month and samples investigated at 30 °C for 2 weeks, the decrease ranges of the F4 and F5 formulation samples were both larger than those of the remaining samples, and the decrease range of F9 was smaller than those of the remaining samples, and the decrease ranges of the remaining samples were close. According to the sample data investigated at 2 - 8 °C for 1 month, the main peak purity of the F5 sample decreased by 2.1%, and there was no significant change in the main peak purity of the remaining formulation samples. According to the data of samples subjected to 5 cycles of freeze-thaw, the main peak purity of the F8 sample decreased by 10.3%, and there was no significant change in the main peak purity of the remaining samples. [Table 12] 2.6 Results of Purity (SDS-PAGE) in the Screening Experiment of Auxiliary Materials and Surfactants

[0084] The SDS-PAGE data are summarized in Table 13. According to the reducing SDS-PAGE data, there was no significant difference in the purity data among different samples. According to the non-reducing SDS-PAGE data, the purity of the F9 sample was better than that of the remaining samples, and the purity data of the remaining samples were close.

Table 13

[0085] According to the measurement results of appearance and insoluble particles, polysorbate 80 is more advantageous in avoiding the generation of particles than polysorbate 20, and the glycine buffer system had a weaker stabilizing effect on proteins than the histidine buffer system.

[0086] According to the SEC data, the protective effect of mannose on the IM001 protein was weaker than that of sucrose and trehalose. According to the DSC and DLS results, the IM001 molecules were prone to chain cleavage and polymerization under low temperature conditions (40 - 50°C). The F2 and F3 formulation samples had similar data under each consideration condition, indicating that the stabilizing effects of sucrose and trehalose on proteins were similar. The F4 and F5 formulation samples had slightly lower main peak purity than the remaining samples under each consideration condition, indicating that the histidine buffer system at pH 8.0 was superior to the glycine and Tris buffer systems. The F2 and F6 formulation samples had similar data under each consideration condition, indicating that there was no significant difference between the addition of 1 mM methionine in the F6 formulation and the absence of methionine in the F2 formulation. The F2 and F7 formulation samples had similar data under each consideration condition, indicating that the stability of the combination of 4% mannitol and 2% sucrose in the F7 formulation was similar to that of the F2 formulation containing 250 mM sucrose. The F9 formulation sample had a higher main peak purity than F2 under each consideration condition, indicating that a protein concentration of 15 mg / mL in the aqueous formulation was more advantageous for the stability of the IM001 protein than 30 mg / mL.

[0087] According to the reduced SDS-PAGE data, there was no significant difference in the purity data between different samples. According to the non-reduced SDS-PAGE data, the purity of the F9 sample was better than that of the remaining samples, and the purity data of the remaining samples were similar. Since the F2 and F9 formulations had the same other compositions except for different concentrations, it was shown that a protein concentration of 15 mg / mL was more favorable for the stability of IM001 protein than 30 mg / mL for the aqueous formulation. Example 3

[0088] The research objective of this example is to screen high-concentration lyophilized formulation prescriptions.

[0089] According to the results of screening the aqueous formulation, the concentration of IM001 protein increased up to 30 mg / mL, and at temperatures of 25°C or higher, the purity showed a significant tendency to decrease as the standing time increased. Therefore, the formulation design of the lyophilized preparation was carried out, and the formulation is shown in Table 14. The IM001 stock solution was exchanged into a 25 mM histidine buffer system (pH 8.0), and different auxiliary material mother liquors and surfactant mother liquors were added based on the experimental plan, and the protein content was adjusted. Different types of sugar mother liquors and surfactant mother liquors were added to adjust the protein content up to 30 mg / mL. The five prepared formulations were filtered using a 0.22 μm PVDF membrane in a biosafety cabinet respectively, and then 5 mL of the drug solution was taken with a pipette into a 20R vial that had been washed and sterilized, immediately stoppered, capped, and labeled. Then, lyophilization was carried out to start the stability study under different conditions, and the specific plan is shown in Table 15.

Table 14

Table 15

[0090] The appearance of the freeze-dried products is shown in Figure 4. After freeze-drying, all formulations appeared as white loose lumps, with F7 (containing 4% mannitol and 2% sucrose) being even looser than the other freeze-dried products. 3.2 Measurement of moisture content of freeze-dried products

[0091] The moisture content of the freeze-dried products is shown in Table 16. The moisture content of the different formulations was less than 3%, which meets the moisture content standard for freeze-dried products, and the specific moisture content is between 1.1% and 1.6%. Among them, the moisture content of F7 is slightly higher at 1.63%. [Table 16] 3.3 Measurement of glass transition temperature (Tg') and freeze-drying collapse temperature (Tc) of freeze-dried formulation frozen solution

[0092] The glass transition temperature (Tg') and freeze-dried collapse temperature (Tc) of the freeze-dried formulation frozen solution are shown in Table 17. The Tg' of the sucrose-containing formulations F2, F16, and F17 are all around -27°C, while the Tg' of the trehalose-containing formulation F7 is slightly higher at -25.9°C, and the lowest Tg' of the 4% mannitol and 2% sucrose-containing formulation F7 is -33.2°C. The Tc of the two formulations F2 and F15 was also measured, and was found to be -23.8°C and -26.7°C, respectively. [Table 17] 3.4 Visual inspection of reconstituted liquid of freeze-dried product

[0093] Based on the difference in mass change of the freeze-dried product before and after freeze-drying, the volume of water added for reconstitution was calculated, and the freeze-dried product was reconstituted with water. After reconstitution, the appearance of the reconstituted solution was observed, and the results are shown in Table 18. After reconstitution, all the samples, including T0, and the samples placed under accelerated conditions (25°C) and high-temperature conditions (40°C), were pale yellow with a slight opalescence and contained no visible particles. [Table 18] 3.5 Determination of pH, protein concentration and osmotic pressure of reconstituted solution of lyophilized product

[0094] The pH, protein concentration and osmotic pressure of the solutions after reconstitution of the freeze-dried products of different formulations are summarized in Table 19. The data show that there were no significant changes in the pH, protein concentration and osmotic pressure of the five formulation samples after they were left under accelerated conditions (25°C) and high temperature conditions (40°C). [Table 19] 3.6 Detection of insoluble particles (MFI) in the reconstituted solution of lyophilized products

[0095] The results of insoluble particle detection (MFI) of the reconstituted freeze-dried product are summarized in Table 20. There was no significant difference in the number of insoluble particles for the different formulations, and the number of insoluble particles after reconstitution did not show a clear increase compared to the TO sample after storage at 25°C for 1 month or at 40°C for 2 months. [Table 20] 3.7 Purity of reconstituted solution of lyophilized product (SEC-HPLC)

[0096] The SEC-HPLC results are shown in Table 21. All formulations were stored at accelerated (25°C) and elevated (40°C) temperatures for 2 weeks to 3 months. The SEC purity of F7 decreased slightly, but the SEC purity of the remaining formulations remained essentially unchanged, demonstrating significantly improved stability of the lyophilized products compared to the liquid formulations. The stability of F7 (containing 4% mannitol and 2% sucrose) was poorer than the other formulations, possibly due to the crystallization of mannitol after annealing and the loss of the protective effect of sugar on proteins. The SEC main peak content of the remaining formulations remained essentially unchanged, demonstrating good stability. [Table 21] 3.8 Summary of the lyophilized formulation screening experiment

[0097] Among the five lyophilized products investigated, all had good appearance and were white, porous lumps. The measured moisture content was 1.1 - 1.6%, all less than 3%, and the moisture content was qualified. They were left standing for 2 weeks to 3 months under accelerated conditions (25°C) and high-temperature conditions (40°C), and the reconstituted solutions were all light yellow, slightly opalescent, and had no visible particles. The Tg’ of F7 (containing 4% mannitol and 2% sucrose) was -33.2°C, and the Tg’ of the remaining formulations was about -26 to -27°C. There was no significant difference in the MFI results among different formulations. According to the SEC results, all the investigated formulations except F7 had good stability after lyophilization. They were left standing for 3 months under accelerated conditions (25°C) and 2 months under high-temperature conditions (40°C), and there was no obvious decrease in their main peaks. The SEC main peak of the F7 formulation (containing 4% mannitol and 2% sucrose) decreased more than that of other formulations, and its stability was worse. Summary

[0098] According to the buffer system screening results, the stability of the pH 8.0 His buffer salt (formulation pH 7.6) system was the best.

[0099] According to the co-formulant and surfactant screening results, the stability of the pH 8.0 histidine buffer system was stronger than that of the glycine and Tris buffer systems. The protective effects of sucrose and trehalose were similar, superior to that of mannitol, and the stability of the 15 mg / mL protein concentration was superior to that of the 30 mg / mL system. Among them, the stability of the aqueous formulation of the F9 formulation (15 mg / mL protein, 25 mM His, 250 mM sucrose, 0.02% PS80, pH 7.6) was the best.

[0100] The results of the freeze-dried formulation screening experiment showed that the stability of the freeze-dried product was significantly improved compared to the solution formulation. Among them, the expression of F7 formulation (30 mg / mL protein, 25 mM His pH 8.0, 4% mannitol, 2% sucrose, 0.02% PS80) was slightly poor, but the remaining formulations all showed excellent stability.

[0101] Based on the above experimental results, the F15 formulation (30 mg / mL protein, 25 mM His, 250 mM Trehalose, 0.02% PS80, pH 7.6) was selected as the freeze-dried formulation of IM001 protein.

[0102] As described above, the present invention is merely a preferred embodiment, and does not necessarily limit the present invention in any form or substance. It should be pointed out that even those skilled in the art can make some improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Those skilled in the art will appreciate that any slight changes, modifications, and equivalent changes made by those skilled in the art using the technical content disclosed above without departing from the spirit and scope of the present invention are all equivalent embodiments of the present invention, and at the same time, any changes, modifications, and evolutions made to the above embodiments based on the essential technology of the present invention are all within the scope of the technical solution of the present invention. In certain embodiments, for example, the following items are provided. (Item 1) An aqueous formulation comprising a fusion protein containing two monomers having an amino acid sequence of SEQ ID NO: 1, linked by a plurality of disulfide bonds, 20 - 30 mM histidine salt, 220 - 280 mM trehalose, and 0.01 (w / v)% - 0.03 (w / v)% polysorbate 80, and having a pH of 7.0 - 8.2. (Item 2) The aqueous formulation according to Item 1, wherein the pH of the aqueous formulation is 7.4 - 7.8. (Item 3) The aqueous formulation according to Item 2, wherein the pH of the aqueous formulation is 7.6. (Item 4) The aqueous formulation according to Item 1, wherein the aqueous formulation contains 24 - 26 mM histidine salt. (Item 5) The aqueous formulation according to Item 4, wherein the aqueous formulation contains 25 mM histidine salt. (Item 6) The aqueous formulation according to Item 1, wherein the aqueous formulation contains 240 - 260 mM trehalose. (Item 7) The aqueous formulation according to Item 6, wherein the aqueous formulation contains 250 mM trehalose. (Item 8) The aqueous formulation according to Item 1, wherein the aqueous formulation contains 0.015 (w / v)% - 0.025 (w / v)% polysorbate 80. (Item 9) The aqueous formulation according to Item 8, wherein the aqueous formulation contains 0.02 (w / v)% polysorbate 80. (Item 10) The aqueous formulation according to Item 1, further comprising an isotonic agent, a surfactant, an antioxidant, a preservative, or a mixture thereof. (Item 11) The aqueous formulation according to Item 1, wherein each fusion protein molecule contains 6 or fewer galactose-α-1,3-galactose. (Item 12) The aqueous formulation according to Item 11, wherein each fusion protein molecule contains 3 or fewer galactose-α-1,3-galactose. (Item 13) The aqueous formulation according to Item 11, wherein the fusion protein contains a glycan, and on average at least 52% of the glycan contains one or more sialic acid residues. (Item 14) The aqueous formulation according to Item 1, containing at least 10 mg / mL of the fusion protein. (Item 15) The aqueous formulation according to Item 14, containing at least 25 mg / mL of the fusion protein. (Item 16) An aqueous formulation according to item 1, comprising at least 25 mg / mL of the fusion protein, 24-26 mM of histidine salt, 240-260 mM of trehalose, and 0.015 (w / v)% - 0.025 (w / v)% of polysorbate 80, and having a pH of 7.4 - 7.8. (Item 17) An aqueous formulation according to item 16, comprising 30 mg / mL of the fusion protein, 25 mM of histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and having a pH of 7.6. (Item 18) An aqueous formulation according to any one of items 1 - 17, which does not contain an amino acid salt other than histidine salt higher than 10 mM, or preferably, does not contain other amino acid salts. (Item 19) An aqueous formulation according to item 18, which does not contain a sugar other than trehalose higher than 10 mM, or preferably, does not contain a sugar other than trehalose. (Item 20) An aqueous formulation according to item 19, consisting of 30 mg / mL of the fusion protein, 25 mM of histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and having a pH of 7.6. (Item 21) An aqueous formulation according to any one of items 1 - 20, which is for treating an inflammatory disease or an IL-6-mediated disorder in humans. (Item 22) The inflammatory disease or IL-6-mediated disorder is inflammatory bowel disease, and preferably, here, the treatment induces remission of inflammatory bowel disease. An aqueous formulation for the use according to item 21. (Item 23) The inflammatory bowel disease is Crohn's disease or ulcerative colitis, and preferably, here, the treatment maintains remission of inflammatory bowel disease. An aqueous formulation for the use according to item 21. (Item 24) The inflammatory disease or IL-6-mediated disorder is rheumatoid arthritis, psoriasis, uveitis or atherosclerosis. An aqueous formulation for the use according to item 21. (Item 25) The inflammatory disease or IL-6-mediated disorder is colitis not related to inflammatory bowel disease, and preferably, here, the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis, or colitis caused by an allergy affecting the large intestine. An aqueous formulation for the use according to item 21. (Item 26) A dry preparation obtained by freeze-drying the aqueous preparation according to any one of items 1 to 20. (Item 27) A dry formulation that can, upon addition of water, produce the aqueous formulation according to any one of items 1 to 20.

Claims

1. An aqueous formulation comprising a fusion protein containing two monomers with an amino acid sequence of SEQ ID NO: 1, linked by a plurality of disulfide bonds, a 20-30 mM histidine salt, a 220-280 mM trehalose, 0.01 (w / v)% to 0.03 (w / v)% polysorbate 80, and having a pH of 7.0 to 8.

2.

2. The aqueous formulation according to Claim 1, wherein the pH of the aqueous formulation is 7.4 to 7.

8.

3. The aqueous formulation according to Claim 2, wherein the pH of the aqueous formulation is 7.

6.

4. The aqueous formulation according to Claim 1, wherein the aqueous formulation contains 24-26 mM histidine salt.

5. The aqueous formulation according to Claim 4, wherein the aqueous formulation contains 25 mM histidine salt.

6. The aqueous formulation according to Claim 1, wherein the aqueous formulation contains 240-260 mM trehalose.

7. The aqueous formulation according to Claim 6, wherein the aqueous formulation contains 250 mM trehalose.

8. The aqueous formulation according to Claim 1, wherein the aqueous formulation contains 0.015 (w / v)% to 0.025 (w / v)% polysorbate 80.

9. The aqueous formulation according to Claim 8, wherein the aqueous formulation contains 0.02 (w / v)% polysorbate 80.

10. The aqueous formulation according to Claim 1, further comprising an isotonic agent, a surfactant, an antioxidant, a preservative, or a mixture thereof.

11. The aqueous formulation according to Claim 1, wherein each fusion protein molecule contains 6 or fewer galactose-α-1,3-galactose moieties.

12. The aqueous formulation according to Claim 11, wherein each fusion protein molecule contains 3 or fewer galactose-α-1,3-galactose moieties.

13. The aqueous formulation according to Claim 11, wherein the fusion protein contains a glycan, and on average at least 52% of the glycan contains one or more sialic acid residues.

14. The aqueous formulation according to Claim 1, containing at least 10 mg / mL of the fusion protein.

15. The aqueous formulation according to Claim 14, containing at least 25 mg / mL of the fusion protein.

16. The aqueous formulation according to claim 1, comprising at least 25 mg / mL of the fusion protein, 24-26 mM of a histidine salt, 240-260 mM of trehalose, and 0.015 (w / v)% to 0.025 (w / v)% of polysorbate 80, and having a pH of 7.4 to 7.

8.

17. The aqueous formulation according to claim 16, comprising 30 mg / mL of the fusion protein, 25 mM of a histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and having a pH of 7.

6.

18. The aqueous formulation according to any one of claims 1 to 17, which does not contain an amino acid salt other than the histidine salt at a concentration higher than 10 mM, or preferably, does not contain other amino acid salts.

19. The aqueous formulation according to claim 18, which does not contain a sugar other than trehalose at a concentration higher than 10 mM, or preferably, does not contain other sugars than trehalose.

20. The aqueous formulation according to claim 19, consisting of 30 mg / mL of the fusion protein, 25 mM of a histidine salt, 250 mM of trehalose, and 0.02 (w / v)% of polysorbate 80, and having a pH of 7.

6.

21. The aqueous formulation according to any one of claims 1 to 20, which is for treating an inflammatory disease or an IL-6-mediated disorder in a human.

22. The aqueous formulation according to claim 21, wherein the inflammatory disease or IL-6-mediated disorder is an inflammatory bowel disease, and preferably, wherein the treatment induces remission of the inflammatory bowel disease.

23. The aqueous formulation according to claim 22, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis, and preferably, wherein the treatment maintains remission of the inflammatory bowel disease.

24. The aqueous formulation according to claim 21, wherein the inflammatory disease or IL-6-mediated disorder is rheumatoid arthritis, psoriasis, uveitis, or atherosclerosis.

25. The aqueous formulation according to claim 21, wherein the inflammatory disease or IL-6-mediated disorder is a colitis not related to inflammatory bowel disease, and preferably, wherein the colitis is radiation colitis, diverticular colitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis, or colitis caused by an allergy affecting the large intestine.

26. A dry formulation obtained by lyophilizing the aqueous formulation according to any one of claims 1 to 20.

27. A dry formulation capable of producing the aqueous formulation according to any one of claims 1 to 20 when water is added.

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